The mechanisms by which the ultimate pattern of synaptic connections arises in the CNS is one of the great unanswered questions in neurobiology. Some insights into this question may be gleaned from the structure of synaptic junctions. Each excitatory CNS synapse is an asymmetric cell-cell junction, with very different sets of molecules clustered on the respective sides of the junction. How the different sets of molecules target to synaptic membranes and remain in direct apposition to each other is still not completely understood but trans-synaptic interactions must have an important role in this process and are also probably involved in the specification of synaptic connectivity. We hypothesize that the synaptic cleft contains a number of uncharacterized molecules important for trans-synaptic interactions. Identification of such molecules will shed new light on the role of pre-to-postsynaptic recognition in the maintenance of synaptic architecture and in the generation of connectivity. In this R21 proposal, we will use a targeted biochemical approach to identify the molecules that mediate trans-synaptic interactions, made possible by the PIs preliminary studies in which intact synaptic junctions were isolated from the CNS, preserving trans-synaptic molecules and their interactions. In Aim 1, we will recover synaptic cleft proteins on isolated synaptic membranes by utilizing cell surface biotinylation and immobilized lectins. Recovered candidate synaptic cleft proteins will be electrophoresed and identified by mass spectrometry. In Aim 2, we will develop antibodies to identified candidate synaptic cleft proteins for use at the light and EM levels to evaluate the synaptic localization of potential cleft proteins. Upon completion of these Aims, we expect to have in hand reagents corresponding to some of the most prominent synaptic cleft molecules. We will use these reagents to examine the role of cell-cell interactions in the initiation, maturation and plasticity of synaptic junctions and in the recruitment of other critical molecules to synaptic membranes.